副猪嗜血杆菌OMP P2基因PCR检测方法的建立与应用

Application and development of PCR assay Amplifying OMP P2 gene of Haemophilus parasuis

通过反应条件的优化、特异性和敏感性的测定,建立了一种快速、简便、准确的PCR方法,用于检测副猪嗜血杆菌(HPS)OMP P2基因。PCR扩增片段大小为1 080 bp左右,最小检测量为1 000个HPS。与胸膜肺炎放线杆菌、多杀性巴氏杆菌、大肠杆菌、肠沙门菌、金黄色葡萄球菌和链球菌无特异性反应。用该法检测人工感染猪的肺脏、心脏和肾脏抽提的DNA,结果为阳性;检测肝脏、脾脏、脑组织样品抽提的DNA,结果为阴性。用该法检测人工感染发病仔猪及自然感染仔猪肺组织,并结合细菌分离培养,证明以肺脏组织为病料,通过细菌分离培养,再用PCR法鉴定分离菌体,可有效地用于HPS的特异诊断。检测116份来自发病猪场的患病仔猪肺脏病料,其中有110份为阳性,与16S rRNA PCR检测结果一致,表明HPS所致发的Glsser's病已在我国广泛发生和流行。

A PCR assay for amplifying OMP P2 gene of Haemophilus parasuis（HPS） was developed for quick and accurate detection of HPS.The size of the amplified PCR product was approximately 1 080 bp.The required minimum dose of HPS was 1000 bacteria.The specificity of the PCR was tested with Actinobacillus pleuropnemoniae,Pasteurella multocida,E coli,Salmonella enterica,Staphylococcus aureus and Streptococcus,the PCR test developed in the present work did not recognize any members of these species.The positive results were obtained by amplifying the extracted DNA from the artificial challenged piglets,while the negative results from the natural infection.Twelve clinical samples（6 from experimentally infected piglets with HPS isolates and 6 from naturally infected piglets） were tested by PCR.The results showed that the PCR assay could be used to detect HPS from lung sample.When the assay was used to detect the sick piglets lung sample from 116 pig farm,the results showed that the 110 samples of which were positive to HPS,with the same results as 16S rRNA PCR test.It indicated that the P2-PCR assay could be used to specially detect HPS,and that its resultant disease,the Glsser＇s disease,has been prevailled in China.